Binding agent for growth factor

ABSTRACT

A binding agent for a growth factor selected from the group consisting of transforming growth factor-β (TGF-β) and platelet-derived growth factor (PDGF), comprising a protein-bound polysaccharide which is obtained from a fungus belonging to Coriolus, and contains about 18 to 38% by weight of proteins, and a pharmaceutically acceptable carrier or diluent is disclosed.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a binding agent for particular growthfactors, namely, transforming growth factor-β (TGF-β) andplatelet-derived growth factor (PDGF). The binding agent of the presentinvention selectively binds to the particular growth factors to inhibittheir biological activities. Therefore, the binding agent of the presentinvention can be used for treatment or prevention of various diseasesdirectly or indirectly caused by the particular growth factors.

2. Description of the Related Art

Various factors which are produced in a living body and act on aparticular cell to promote its growth have been found, and thestructures and the functions thereof have been elucidated. It has beendiscovered that some growth factors exhibit not only the promotion ofthe growth, but also the various biological activities, and that theoverproduction and/or abnormal metabolism of such factors are involvedin various diseases.

Transforming growth factor-β (TGF-β) and platelet-derived growth factor(PDGF) belong to such a growth factor. TGF-β was first found as a factorto promote growth of a rat fibroblast. Thereafter, it has been foundthat TGF-β inhibits growth of a lot of cells, strongly suppressesimmunological activity, and increases extracellular matrix. It issuggested that the overproduction and/or abnormal metabolism of TGF-βare involved in various diseases and symptoms of immunosuppression in acancer patient or the like, fibroid lung, hepatic fibrosis,glomerulonephritis, scleroderma, or the like. Further, PDGF acts onsmooth muscle cells, fibroblasts, nerve gliacytes or the like to promotetheir growth. The relationship between PDGF and arterial sclerosiscaused by wandering and growth of vascular smooth muscle cells attractsattention.

It would be possible to use an antibody which specifically binds toTGF-β and/or PDGF to inhibit their functions, as a means of inhibitingthe functions of the growth factors. In fact, the attempts to use suchan antibody were actually made. Nevertheless, when the antibody isadministered, there exist many problems to be solved, for example,prevention of allergic reaction caused by the administration, orprevention of the activity reduction by the production of antibodies toneutralize the administered antibody. Therefore, it is desired todevelop an agent which inhibits the function of the growth factors, andcan be safely administered.

SUMMARY OF THE INVENTION

The inventors of the present invention engaged in intensive studies tosolve the above problems, and as a result, found that the particularprotein-binding polysaccharide which is obtained from Basidiomycetes,and which has been clinically used as a carcinostatic agent and provedto hardly produce side-effects, binds to the above growth factors toinhibit the functions thereof. The present invention is based on thefindings.

Accordingly, the object of the present invention is to provide a bindingagent for the above growth factors.

Other objects and advantages will be apparent from the followingdescription.

In accordance with the present invention, there is provided a bindingagent for a growth factor selected from the group consisting oftransforming growth factor-β (TGF-β) and platelet-derived growth factor(PDGF), comprising a protein-bound polysaccharide which is obtained froma fungus belonging to Coriolus, and contains about 18 to 38% by weightof proteins.

BRIEF DESCRIPTION DRAWINGS

FIG. 1 is a graph showing the results of an enzyme-immunoassay () andradio-immunoassay (◯) of the binding of TGF-β and PSK by the reactiontherebetween, as carried out in Example 1.

FIG. 2 is a graph showing the results of the control experiments usingonly ¹²⁵ I labeled TGF-β₁, in the experiments wherein ¹²⁵ I labeledTGF-β₁ was reacted with PSK, the reaction mixture was applied on a gelfiltration chromatography column, and the radioactivities in fractionswere measured as carried out in Example 2.

FIG. 3 is a graph showing the results of the case that ¹²⁵ I labeledTGF-β₁ and PSK were added, in the experiments wherein ¹²⁵ I labeledTGF-β₁ was reacted with PSK, the reaction mixture was applied on a gelfiltration chromatography column, and the radioactivities in fractionswere measured as carried out in Example 2.

FIG. 4 is a graph showing the restoring effect of PSK to the growthinhibition by TGF-β₁, in the experiments wherein PSK and TGF-β₁ wereadded to the in vitro growing system of the fetal mink lung cell lineMv1Lu, as carried out in Example 3.

FIG. 5 is a graph showing a dose-dependency of the restoring effect ofPSK to the growth inhibition by TGF-β₁, in the experiments wherein PSKand TGF-β₁ were added to the in vitro growing system of the fetal minklung cell line Mv1Lu, as carried out in Example 3.

FIG. 6 is a graph showing the restoring effect of PSK to the growthinhibition by TGF-β₁, in the experiments wherein PSK and TGF-β₁ wereadded to the in vitro growing system of the mouse T cell line CTLL-2, ascarried out in Example 3.

FIG. 7 is a graph showing a dose-dependency of the restoring effect ofPSK to the growth inhibition by TGF-β₁, in the experiments wherein PSKand TGF-β₁ were added to the in vitro growing system of the mouse T cellline CTLL-2, as carried out in Example 3.

FIG. 8 is a graph showing a dose-dependency of the TGF-β₁ binding to thefetal mink lung cell line Mv1Lu, in the experiments wherein the ¹²⁵ Ilabeled TGF-β₁ was added to the in vitro binding assay system of thecell line Mv1Lu, as carried out in Example 4.

FIG. 9 is a graph showing a dose-dependency of the TGF-β₁ binding to themouse T cell line CTLL-2, in the experiments wherein the ¹²⁵ I labeledTGF-β₁ was added to the in vitro binding assay system of the cell lineCTLL-2, as carried out in Example 4.

FIG. 10 is a graph showing a dose-dependency of the cell bindinginhibitory by PSK, in the experiments wherein the ¹²⁵ I labeled TGF-β₁and PSK were added to the in vitro binding assay system of the fetalmink lung cell line Mv1Lu, as carried out in Example 4.

FIG. 11 is a graph showing a dose-dependency of the cell bindinginhibitory by PSK, in the experiments wherein the ¹²⁵ I labeled TGF-β₁and PSK were added to the in vitro binding system of the mouse T cellline CTLL-2, as carried out in Example 4.

FIG. 12 is a graph showing the effect by the differences of the tumorsizes, when TGF-β₁ and/or PSK were administered to the leukemiccells-implanted mice, as carried out in Example 5.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The protein-bound polysaccharide used as an active ingredient in thebinding agent of the present invention is disclosed in, for example,Japanese Examined Patent Publications (Kokai) No. 46-17149, No.51-36322, No. 56-14274, No. 56-14275, and No. 56-14276. Theprotein-bound polysaccharide is an extract from mycelia, broth, or fruitbodies that is obtained by culturing the fungus belonging to Coriolus,which belongs to Basidiomycetes. The protein-bound polysaccharidecontains about 18 to 38% by weight of proteins, and has a molecularweight of not less than 5,000, preferably 5000 to 3000,000. Further, theprotein-bound polysaccharide is described in "Saikin no Shinyaku (RecentNew Medicines)", Vol. 28, 14-16, 1977; and Vol. 29, 96-101, 1978; and"Iyakuhin Yoran (Handbook of Medicines)", 6th Ed., 1346, 1979, publishedby Yakuji Jiho Publishing Co.

A typical example of the protein-bound polysaccharide is called PSK,which is commercially available as "KRESTIN" (trade mark) from SankyoCo., Ltd.

The properties of PSK are as follows:

PSK may be prepared by extracting the mycelia of Coriolus versicolor(Fr.) Quel FERM-P2412 (ATCC20547)! with hot water, purifying and thendrying the extract. The average molecular weight (ultracentrifugalmethod) is about 100,000. The sugar portion in the major fractions isβ-D-glucan. The glucan has a branched structure containing 1→3, 1→4 and1→6 bonds, and mainly comprises glucose (74.6% by weight) and mannose(15.5% by weight). In the amino acid composition of the proteins, thereare many acidic amino acids, such as aspartic and glutamic acids, manyneutral amino acids, such as valine and leucine, but a few basic aminoacids, such as lysine and arginine. PSK is soluble in water, but hardlysoluble in methanol, pyridine, chloroform, benzene or hexane. PSK beginsto be gradually decomposed at about 120° C.

The protein-bound polysaccharide is extremely safe, because of very lowtoxicity and few side-effects. The acute toxicity of PSK is shown in thefollowing Table 1.

                  TABLE 1                                                         ______________________________________                                                          LD.sub.50 (mg/kg)                                           Animal sp.                                                                              Route         Male    Female                                        ______________________________________                                        Mice      per os        >20000  >20000                                        Rats      per os        >20000  >20000                                        ______________________________________                                    

The acute toxicity shown in Table 1 was determined by the followingmethod. The 4- or 5-week-old ICR-JCL mice (21 to 24 g) and the 4- or5-week-old Donryu rats (100 to 150 g) were used. The agent was orallyadministered as shown in Table 1. The protein-bound polysaccharide wasdissolved in physiological saline and the solution was administered.Death and body weight were observed for 7 days. Thereafter, the animalswere sacrificed for autopsy. No death was observed in mice and rats evenby the maximum administrable doses, and thus, the determination of LD₅₀was substantially impossible.

The protein-bound polysaccharide used as an active ingredient in thebinding agent of the present invention selectively binds to TGF-β andPDGF, but does not bind to various cytokines including growth factorsother than TGF-β and PDGF. For example, as shown in Examples 1 and 2,the protein-bound polysaccharide does not bind to epidermal growthfactor (EGF); interleukin (IL)-1α, IL-1β, IL-2, IL-3, IL-4, IL-6, IL-7,IL-8, or IL-10; tumor necrosis factor (TNF)-α or TNF-β; interferon(IFN)-α, IFN-β, or IFN-γ; granulocyte colony-stimulating factor (G-CSF);or granulocyte-macrophage colony-stimulating factor (GM-CSF).

The TGF-β which binds to the protein-bound polysaccharide is notlimited, but includes, for example, TGF-β₁, TGF-β₂, TGF-β₃, TGF-β₄, andTGF-β₅.

The protein-bound polysaccharide used as an active ingredient in thebinding agent of the present invention inhibits the physiologicalactivity of TGF-β and PDGF by selectively binding thereto. This will beconcretely demonstrated in Examples 3 and 4 (in vitro) and Example 5 (invivo), respectively. More particularly, when TGF-β is added to an invitro growth reaction culture of cells highly sensitive to TGF-β (i.e.,the fetal mink lung cell line Mv1Lu and the mouse T cell line CTLL-2),the growth of the cells is inhibited. If the protein-boundpolysaccharide is added in advance to the in vitro growth reactionculture, the inhibitory action of TGF-β is reduced. The binding of TGF-βto the cells is also inhibited. Further, in the in vivo experiment ofmice to which leukemic cells are implanted, the tumor growth promoted byadministering TGF-β is inhibited by administering PSK.

As above, the protein-bound polysaccharide selectively binds only to theparticular growth factors, i.e., TGF-β and PDGF, and thus, the functionof the protein-bound polysaccharide is limited to the inhibition of thephysiological activities of bound TGF-β and PDGF only. Therefore, it isbelieved that no side-effect caused by any inhibition of functions ofother growth factors is produced. Further, the protein-boundpolysaccharide has extremely low toxicity, and has been clinicallyproved to hardly produce side-effect. Thus, it may be safelyadministered for a long period of time.

When the binding agent of the present invention is administered to ananimal including human as an inhibitor of TGF-β and/or PDGF, the bindingagent may be formulated into a composition for various routes, such as acomposition for oral administration, injections for subcutaneous,intravenous, or intramuscular administration, or a suppository forrectal administration. The composition for oral administration is, forexample, tablet, granule, powder, or capsule. The composition maycontain a binder, excipient, lubricant, disintegrating agent, or wettingagent. The oral liquid composition may be in the form of mixture forinternal use, shaking mixture, suspension, emulsion, or syrup, or in theform of a dry product which should be re-dissolved when used. Further,the above oral liquid composition may contain an additive orpreservating agent. A parenteral composition, such as injection,suppository, or ointment, may contain an additive, such as a stabilizingagent, buffer, preservating agent, or isotonicity. The composition maybe in the form of an aqueous solution, suspension, solution, or emulsionin oily or aqueous vehicle. Alternatively, the active ingredient may bein the form of powder which should be re-dissolved in a suitablevehicle, such as sterilized water without a pyrogenic substance, whenused.

The dose of the binding agent may vary with administration routes, ages,individual differences, or symptoms, but in general, 0.5 to 1000 mg/kgweight/day. In an oral administration, a dose of 20 to 1000 mg may beadministered once or divided into two or three.

EXAMPLES

The present invention will now be further illustrated by, but is nomeans limited to, the following Examples.

Example 1

To a vial containing 1 μg of lyophilized human TGF-β₁ (Takara Shuzo), 5mM HCl (100 μl) containing bovine serum albumin (2 mg/ml) was added todissolve human TGF-β₁. Then, 9.9 ml of phosphate buffered physiologicalsaline (pH 7.4) containing bovine serum albumin (2 mg/ml) was addedthereto to prepare a TGF-β₁ solution (100 ng/ml).

Further, PSK (Trademark "Krestin"; Sankyo) was dissolved in phosphatebuffered physiological saline (pH 7.4) containing bovine serum albumin(2 mg/ml) to prepare PSK solutions containing 0.1 μg/ml, 0.5 μg/ml, 1.0μg/ml, 5.0 μg/ml, 10 μg/ml, 50 μg/ml, 100 μg/ml, 500 μg/ml or 1000 μg/mlof PSK.

The above TGF-β₁ (100 ng/ml) solution (100 μl) and the PSK (0.1 to 1000μg/ml) solution (100 μl) were mixed in a test tube, and reaction wasperformed at 22° C. for 3 hours.

After the reaction was completed, the TGF-β₁ content in the mixedsolution was measured by a commercially available Enzyme Immunoassay kit(TGF-β₁ ELISA System; Amersham Japan). The results are shown by closedcircle () in FIG. 1. Further, the results by radioimmunoassay using ¹²⁵I labeled anti-TGF-β₁ antibodies are shown by open circle (◯) in FIG. 1.The binding ratio (%) in the axis of FIG. 1 is calculated from theequation (I):

    Binding ratio (%)=(Cc-Cp)/Cc×100                     (I)

wherein Cc is a measured content of TGF-β₁ in control groups, and Cp isa measured content of TGF-β₁ in PSK-addition groups.

As apparent from FIG. 1, the TGF-β₁ content in PSK-addition groups isdose-dependently lowered in comparison with that in control groups.Thus, it is manifest that PSK binds to the epitope of TGF-β₁.

The binding activities of PSK and various cytokines and growth factorsother than TGF-β were measured, using commercially available assay kit.The results in Table 2 clearly show that PDGF binds to PSK, as is thecase with TGF-β, but PSK does not bind to other cytokines or growthfactors. The binding ratio (%) in Table 2 were calculated from theequation (I).

                  TABLE 2                                                         ______________________________________                                        Binding ratio (%)                                                             Growth factor/Cytokine                                                                        PSK concentration (μg/ml)                                  (available from)                                                                              1         10      100                                         ______________________________________                                        rhTGF-β.sub.1                                                                        (1)     32        68    96                                        nhTGF-β.sub.1                                                                        (1)     20        67    83                                        rhTGF-β.sub.1                                                                        (2)     0         0     0                                         rhEGF       (3)     0         0     0                                         rhPDGF      (3)     11        26    54                                        rhIL-1α                                                                             (2)     0         0     0                                         rhIL-1β                                                                              (3)     0         0     0                                         rhIL-2      (3)     0         0     0                                         rhIL-3      (3)     0         0     0                                         rhIL-4      (2)     0         0     0                                         rhIL-6      (2)     0         0     0                                         rhIL-7      (2)     0         0     0                                         rhIL-8      (2)     0         0     0                                         rmIL-10     (2)     0         0     0                                         rhTNF-α                                                                             (2)     0         0     0                                         rhTNF-β                                                                              (3)     0         0     0                                         rhIFN-β                                                                              (2)     0         0     0                                         rhIFN-γ                                                                             (4)     0         0     0                                         rhG-CSF     (2)     0         0     0                                         rhGM-CSF    (5)     0         0     0                                         ______________________________________                                         n = natural; r = recombinant; h = human; m = mouse                            Makers of enzyme immunoassay kit                                              (1) = Amersham Japan                                                          (2) = R & D (USA)                                                             (3) = kit from Otsuka Seiyaku                                                 (4) = kit from TorayFuji                                                      (5) = Oncogene Science                                                   

Example 2

To a vial containing 370 kBq (10 μCi) of ¹²⁵ I labeled human recombinantTGF-β₁ (74-167 TBq/mmol; Daiichi Kagaku Yakuhin), 1.0 ml of phosphatebuffered physiological saline (pH 7.4) containing bovine serum albumin(2 mg/ml) was added to prepare a solution of ¹²⁵ I labeled TGF-β₁.

Then, PSK (Sankyo) was dissolved in phosphate buffered physiologicalsaline (pH 7.4) containing bovine serum albumin (2 mg/ml) to prepare asolution of PSK (200 μg/ml).

In a test tube, 500 μl of the ¹²⁵ I labeled TGF-β₁ solution and 500 μlof the solution of PSK (200 μg/ml) or phosphate buffered physiologicalsaline (pH 7.4) containing bovine serum albumin (2 mg/ml). Then, thereaction was performed at 22° C. for 3 hours.

After the reaction was completed, the reaction mixture was applied on agel filtration column (diameter=1 cm; height=50 cm) filled withBio-GelP-60 (Nihon Bio-Rad Laboratories) to carry out gel filtrationchromatography. As an eluting solution, phosphate buffered physiologicalsaline (pH 7.4) containing 0.1% bovine serum albumin was used. Each ofthe eluates was collected in 1 ml portions by a fraction collector, andthe radioactivities were measured by a gamma counter.

The results were shown in FIG. 2 (groups to which only TGF-β₁ was added)and FIG. 3 (groups to which PSK and TGF-β₁ were added). In the controlgroups (FIG. 2), i.e., the groups to which only TGF-β₁ was added, theradioactivities were eluated mainly in fraction number 40 (molecularweight=about 25000, i.e., the molecular weight of the active TGF-β). Inthe groups wherein PSK was reacted (FIG. 3), the radioactivities wereobserved in the fraction numbers 10 to 18 (eluating position of PSK).Therefore, the results of gel filtration chromatography show the bindingof PSK and TGF-β.

Example 3

The fetal mink lung cell line Mv1Lu (Dainippon Pharmaceutical) or themouse T cell line CTLL-2 (Rikagaku Kenkyusho) was suspended in theculture medium as mentioned below so that the concentration became 5×10⁵cells/ml, and 100 μl of the suspension was poured in each well of96-well culture plate (Falcon 3072; Becton-Dickinson Labware, N.J.,USA). Then, a TGF-β₁ solution prepared from the lyophilized human TGF-β₁(Takara Shuzo) as in Example 1 was added to each well so that theconcentration thereof became 50 ng/ml. The cells were cultured in a 5%CO₂ incubator at 37° C. for 48 hours. As the culture medium for the cellline Mv1Lu, Eagle's minimum essential medium containing 10% fetal bovineserum, 2 mM glutamine and 0.1 mM non-essential amino acids was used. Asthe culture medium for the cell line CTLL-2, the RPMI 1640 mediumcontaining 10% fetal bovine serum, 20 units/ml mouse interleukin-2, and2 mM glutamine was used. At 4 hours before the end of the cultivation,15 μl of MTT reagent i.e., a solution prepared by dissolving3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide inphosphate buffered physiological saline so that the concentration became5 mg/ml! was poured to each well. After the cultivation was completed,formazan formed in cells was dissolved in 200 μl of 40 mM HCl acidicisopropanol, and the absorbance at 570 nm was measured by a plate reader(control culture test).

Further, the experiments wherein a PSK solution prepared as in Example 1was added to said culture system were carried out.

In the test (A) for the fetal mink lung cell line Mv1Lu, the PSKsolution was added to each well to which the Mv1Lu suspension had beenpoured as in the control culture test, so that the concentration of PSKbecame 100 μg/ml, then TGF-β₁ was added so that the concentration became50 ng/ml, and thereafter the procedure of the control culture test wasrepeated. In the test (B) for the fetal mink lung cell line Mv1Lu, afterthe PSK solution was added as in the test (A), the cells were thoroughlywashed with the culture medium to remove unbound PSK, then TGF-β₁ wasadded so that the concentration became 50 ng/ml, and thereafter theprocedure of the control culture test was repeated. The results areshown in FIG. 4. In FIG. 4, "PSK(-)" is the results of the controlculture test wherein PSK was not added, "A" is the results of the test(A) wherein after PSK was added, TGF-β₁ was added without washing, and"B" is the results of the test (B) wherein after PSK was added and theunbound PSK was removed by washing, TGF-β₁ was added.

Further, the procedure of the test (A) was repeated, except that PSK wasadded so that the concentration thereof became 1 μg/ml, 10 μg/ml, and100 μg/ml. The results are shown in FIG. 5.

In the test (A) for the mouse T cell line CTLL-2, the PSK solution wasadded to each well to which the CTLL-2 suspension had been poured as inthe control culture test, so that the concentration of PSK became 100μg/ml, then TGF-β₁ was added so that the concentration became 50 ng/ml,and thereafter the procedure of the control culture test was repeated.In the test (B) for the mouse T cell line CTLL-2, TGF-β₁ was added toeach well to which the CTLL-2 suspension had been poured as in thecontrol culture test, so that the concentration became 50 ng/ml, thenthe PSK solution was added so that the concentration of PSK became 100μg/ml, and thereafter the procedure of the control culture test wasrepeated. In the test (C) for the mouse T cell line CTLL-2, after thePSK solution was added as in the test (A), the cells were thoroughlywashed with the culture medium to remove unbound PSK, then TGF-β₁ wasadded so that the concentration became 50 ng/ml, and thereafter theprocedure of the control culture test was repeated. The results areshown in FIG. 6. In FIG. 6, "PSK(-)" is the results of the controlculture test wherein PSK was not added, "A" is the results of the test(A) wherein after PSK was added, TGF-β₁ was added without washing, "B"is the results of the test (B) wherein after TGF-β₁ was added, PSK wasadded without washing, and "C" is the results of the test (C) whereinafter PSK was added and the unbound PSK was removed by washing, TGF-β₁was added.

Further, the procedure of the test (A) was repeated, except that PSK wasadded so that the concentration thereof became 1 μg/ml, 10 μg/ml, and100 μg/ml. The results are shown in FIG. 7.

In FIGS. 4 to 7, growth inhibitory ratio (%) was calculated from theequation (II):

    Growth inhibitory ratio (%)=(Ac-Ap)/Ac×100           (II)

wherein Ac is an absorbance at 570 nm of formazan formed in the controlculture test, Ap is an absorbance at 570 nm of formazan formed in thetest wherein TGF-β₁ was added, or TGF-β₁ and PSK were added.

As shown in the above Figures, the growth of the cell lines wasinhibited by TGF-β₁. When PSK was added before adding TGF-β₁, theinhibitory action by TGF-β₁ was lowered in each cell line. Further, thefunction of PSK is dose-dependent. Therefore, it is manifest that thedirect binding of PSK and TGF-β₁ brings about the reduction of TGF-β₁activity.

Example 4

The fetal mink lung cell line Mv1Lu (Dainippon Pharmaceutical) or themouse T cell line CTLL-2 (Rikagaku Kenkyusho) was suspended in theculture medium for the binding tests i.e., Dulbecco's minimum essentialmedium containing 0.1% bovine serum albumin and 25 mM Hepes! so that theconcentration became 1×10⁶ cells/ml. The cells were precultured in a 5%CO₂ incubator at 37° C. for 2 hours. After the cultivation wascompleted, the cells were washed with the culture medium for the bindingtests three times to adjust the cell concentration to 1×10⁶ cells/ml. Tothe resulting cell suspension, a solution of ¹²⁵ I labeled humanrecombinant TGF-β₁ (74-167 TBq/mmol; Daiichi Kagaku Yakuhin) prepared asin Example 2 so that the concentration thereof became 1 ng/ml, 5 ng/ml,10 ng/ml, or 50 ng/ml. The cells were cultured at 22° C. for 3 hours.After the cells were washed by centrifuging, radioactivities bound tocells were measured by a gamma counter. Non-specific bindings of cellswere examined in the presence of non-labeled TGF-β₁. The results areshown in FIG. 8 (the fetal mink lung cell line Mv1Lu) and FIG. 9 (themouse T cell line CTLL-2). As clear from FIGS. 8 and 9, theradioactivity bound to cells were dependent on the concentration ofTGF-β₁ added.

Then, the experiments wherein PSK was added to the above cultivatingsystem were carried out. More particularly, PSK was added to the abovecell suspension so that the concentration of PSK became 1 μg/ml, 10μg/ml, or 100 μg/ml, then TGF-β₁ was added without washing so that theconcentration of TGF-β₁ became 50 ng/ml, and thereafter the aboveprocedure was repeated. The results are shown in FIG. 10 (the fetal minklung cell line Mv1Lu) and FIG. 11 (the mouse T cell line CTLL-2). WhenPSK was added before TGF-β₁ was added, the binding of TGF-β₁ to thecells was inhibited. The function of PSK was dose-dependent. Therefore,it is clear that the action of PSK is caused by the direct binding toTGF-β₁.

In FIGS. 10 and 11, the binding inhibitory ratio (%) was calculated fromthe equation (III):

    Binding inhibitory ratio (%)=(Rc-Rp)/Rc×100          (III)

wherein Rc is the radioactivity (dpm) when PSK is not added, and Rp isthe radioactivity (dpm) when PSK is added.

Example 5

EL4 leukemia cells (distributed from Microbiology department in NationalDefense Medical College, Saitama, Japan) were subcutaneously implantedto 8-week-old female C57BL/6 mice (Charles River Japan) (a groupconsists of 5 mice) in an amount of 1×10⁶ cells. The size of the tumorwas measured by a slide caliper with time. The TGF-β₁ solution preparedas in Example 1 was intraperitoneally administered at a dose of 1 μg pera mouse for 4 days in succession after the implantation of the tumor.Further, the PSK solution prepared as in Example 1 was intraperitoneallyadministered at a dose of 100 mg/kg for 4 days in succession after theimplantation of the tumor. The tumor sizes at the 9th day after theimplantation are shown in FIG. 12, wherein "(-)" means not-administered,and "(+)" means administered. In comparison with the non-treatmentgroup, the growth promotion of the tumor was clearly observed in thegroup wherein TGF-β₁ was administered, but PSK was not administered,whereas in the group wherein TGF-β₁ and PSK were administered, nopromotion was observed and the size was almost same as that in thenon-treatment group. When PSK was added to the group wherein TGF-β₁ wasnot administered, the growth of the tumor was not influenced. Therefore,it is apparent that the function of PSK is caused by the direct actionto TGF-β₁.

As explained above, the protein-bound polysaccharide used in the presentinvention can selectively inhibit only the biological activities ofTGF-β₁ and/or PDGF, without an allergic reaction when an antibodypreparation (anti-TGF-β₁ or PDGF antibody) is administered in a livingbody, or without lowering the activity of the antibody preparation whena neutralizing antibody to the preparation is formed. Further, theprotein-bound polysaccharide used in the present invention has beenclinically used as a carcinostatic agent and proved to hardly produceside-effects.

Although the present invention has been described with reference tospecific embodiments, various changes and modifications obvious to thoseskilled in the art are deemed to be within the sprit, scope, and conceptof the invention.

We claim:
 1. A method for treatment or prevention of a disease orcondition directly or indirectly caused by a growth factor selected fromthe group consisting of transforming growth factor-β (TGF-β) andplatelet-derived growth factor (PDGF), comprising administering to amammal an effective amount of a protein-bound polysaccharide which isobtained from a fungus belonging to Coriolus, and which contains about18 to 38% by weight of protein, said disease or condition being fibroidlung, hepatic fibrosis, glomerulonephritis, or scleroderma.
 2. Themethod according to claim 1, wherein the protein-bound polysaccharide isan extract from mycelia, broth, or fruit bodies obtained by culturing afungus belonging to Coriolus.
 3. The method according to claim 1,wherein the protein-bound polysaccharide is prepared by extractingmycelia of Coriolus versicolor (Fr.) Quel with water, purifying and thendrying a resulting extract.